Urotensin II is a somatosatin-like cyclized peptide that is conserved across many species including fish, frog, mouse, rat, pig, and human (Coulouarn et al., 1999, FEBS. Lett. 457(1): 28-32). G-protein-coupled receptor 14 (GPR14), also known as sensory epithelium neuropeptide-like receptor (SENR), was recently identified as to function as an U-II receptor (UT receptor, Ames et al., 1999, Nature 401(6750): 282-6). Human U-II binds to recombinant human GPR14 with high affinity and the binding is functionally coupled to calcium mobilization.
Emerging roles of U-II in cardiovascular diseases have been implicated (Russell, 2004, Pharmcology & Therapeutics 103: 223-243). Recent evidence suggests that the UT receptor system is up-regulated in multi-organ disease states, such as congestive heart failure (CHF), pulmonary hypertension, and chronic renal failure. A number of non-peptide UT receptor antagonists have been developed with the aim of dampening harmful effects of over-activated UT receptors (see, i.e., Douglas et al, 2004, Trends Pharmacol Sci. 25: 76-85). However, U-II exhibits significant species differences, as well as regional and functional differences between vessels (Douglas et al., 2000, Br. J. Pharmacol. 131(7): 1262-74). Molecules identified as antagonists for the rat receptor can behave as agonists against the monkey receptor (Behm, et al., 2004, European Journal of Pharmacology 492(2-3): 113-116). Thus, it is critical to confirm the effect of a putative drug-like molecule on the biological activities of an endogeneous human UT receptor in a cellular functional assay.
Until recently, there lacked a suitable human cell line for studying the biological activities of an endogeneous UT receptor. U-II binding or calcium mobilization coupled to U-II receptor activation has been detected in very few native cell lines. In 2004, Qi reported that primary human skeletal muscle myoblasts bind U-II. In addition, they reported that the primary human skeletal muscle myoblasts had a slight but significant calcium mobilization in response to U-II (Qi, et al., 2005, Peptides, 26(4):683-690). Douglas et al. (2004, Br. J. Pharmacol. 142(6): 921-32) reported that an appreciable human U-II binding site density was observed in a human skeletal muscle rhabdomyosarcoma cell, SJRH30 (ATCC® Number: CRL-2061™, also named RC13, or RMS 13). However, they also observed that “only ˜10% SJRH30 cells exposed to hU-II responded with an appreciable [Ca2+]i response,” and “the magnitude of the hU-II-induced [Ca2+]i varied significantly between individual cells from ˜10 nM to several hundred nM over baseline.” The calcium mobilization coupled to U-II receptor activation in SJRH30 is not robust enough for high throughput screening of compounds that increase or decrease the biological activity of U-II receptor.
To facilitate the development of new compounds that regulate the biological activity of UT receptor, there is a need to establish a human cell clone that allows robust and simple measurement of the ability of a candidate compound to increase or decrease the biological activity of an endogeneous UT receptor in the cell.